We identified a short peptide SWLRDIWDWICEVLSDFK called C5A, which represents a novel class of microbicidal candidates. C5A neutralizes HIV at an nM-M range without apparent cytotoxicity to human cells. C5A corresponds to a small (18 amino acids) N-terminal region (aa 3-20) of the hepatitis C virus (HCV) nonstructural protein 5A (NS5A) (477 amino acids). The sequence of C5A encompasses the region responsible for the anchoring of NS5A into the ER membrane. Importantly, in contrast to C5A (18 aa), full length NS5A (477 aa) does not inhibit HIV infection. We demonstrated that C5A disrupts the HIV membrane, but preserves the integrity of the cellular plasma membrane. The HIV membrane rupture is specific because C5A does not disturb the integrity of the plasma membrane of human cells even when used at high doses and because it does not inhibit the infection of other enveloped viruses such as influenza and vesicular stomatitis viruses. C5A possesses multiple attractive microbicidal properties: it i) blocks HIV infection of primary targets including T cells, macrophages and dendritic cells; ii) exhibits a broad range of antiviral activity against primary HIV isolates, multi-drug resistant HIV isolates, SIV and SHIV; iii) interrupts an ongoing T cell infection; iv) prevents transmigration of HIV through primary human genital epithelial cells; v) blocks infection of dendritic and Langerhans cells ex vivo (skin tissues); vi) prevents HIV transfer from dendritic and Langerhans cells to T cells ex vivo; vii) is extremely efficacious since less than 15 min of exposure suffices for C5A to neutralize HIV; viii) is potent for a considerable length of time both prior to (at least 1 h) and after (at least 1 h) addition of HIV to cells; ix) is potent at a low pH; x) is soluble in water at inhibitory concentrations; xi) is not toxic to commensal Lactobacilli present in the vaginal tract; xii) exhibits minimal adverse changes, inflammation and toxicity in cervicovaginal tissue in vivo; xiii) is not immunogenic; xiv) does not affect cellular signaling pathways; xv) apparently does not allow viral development resistance; xvi) efficiently blocks HIV infectivity when diluted in genital fluids; and most importantly xvii) vaginal application of C5A offers complete protection against a vaginal viral challenge in the humanized BLT mouse HIV transmission model. Thus, C5A represents the prototype of a new generation of microbicidal agents that may have promise for HIV prevention. In this application, we would like to follow up on these exciting data by fully exploring the possibility that C5A represents a true microbicidal candidate. In the first aim of this application, we propose to conduct a series of experiments aimed at identifying the component of the viral membrane to which C5A binds because the C5A ligand, which resides in the membrane of HIV, represents a potential target for the development of a novel class of anti-HIV therapies with an unusual mechanism of antiviral action. Interestingly, we obtained several lines of evidence that the sphingolipid called dihydrosphingomyelin (DHSM) represents the main target of C5A in the HIV membrane: i) DHSM, incorporated into HIV particles, is specifically pulled down by C5A beads; ii) C5A binds directly to adsorbed DHSM; iii) C5A ruptures liposomes constituted with DHSM; and most importantly iv) pre-incubation of C5A with soluble DHSM prevents HIV rupture by C5A and preserves HIV infectivity. The amphipathic property of C5A, the identity of DHSM as the C5A target in the HIV membrane, and the specific C5A rupture of DHSM-containing liposomes or HIV particles, provide the first hint for the antiviral mechanism of C5A action: C5A, which encompasses the N-terminal region responsible for the anchoring of NS5A into the ER membrane, by binding to DHSM enriched within the HIV membrane, disturbs the integrity of the viral membrane due to its amphipathic nature. In the second aim of this application, we propose to optimize the in vitro potency and in vivo safety of C5A by creating a second generation of peptides using the parental C5A peptide as the archetype. All newly synthesized peptides will be tested in genital fluids for their in vitro microbicidal properties. The most potent compounds among the newly synthesized peptides will be selected. Remarkably, we found that acetylation, amidation and glycosylation of C5A greatly enhanced C5A anti-HIV activities in genital fluids. This is the proof-of-concept for the feasibility of identifying C5A derivates with enhanced anti-HIV activities. In the third aim, the most potent C5A derivates will be assessed for safety and efficacy in the HIV vaginal transmission BLT mouse model. Kinetic administration studies will be executed to determine how long before and/or after the viral challenge C5A precludes HIV transmission. In the fourth aim, safety and efficacy pilot studies will be conducted in another SIV/HIV vaginal transmission animal model: the progesterone-treated macaque model. If similar protective results were obtained using the two transmission models, it would further validate the use of these models for the screening of microbicidal candidates. In addition, protective results would provide proof-of-concept of the usefulness of topically applied microbicides, such as C5A, to prevent genital HIV transmission.